Long-term outcome of insulin pump therapy: reduction of hypoglycaemia and impact on glycaemic control.

AIMS: To determine the long-term outcome of continuous subcutaneous insulin infusion (CSII) in Type 1 diabetes according to Catalan National Health Service indications. METHODS: Retrospective observational study including 178 patients with Type 1 diabetes who started CSII treatment in our centre (2003-2008). All patients were followed in our CSII programme for outpatients for at least 5 years. Data on annual HbA1c levels were collected, and the main indication for starting CSII was analysed. RESULTS: Twenty-seven of 178 patients were excluded because of loss to follow-up or withdrawal from CSII, thus 151 patients (aged 37.4 ± 10.5 years, 64% women) were analysed. The main indications for starting CSII were suboptimal metabolic control (60.9%), severe hypoglycaemia/hypoglycaemia unawareness (25.5%) and others (13.6%). HbA1c was 64 ± 13 mmol/mol (8.0 ± 1.2%) at the start of CSII and 62 ± 13 mmol/mol (7.8 ± 1.2%) after 5 years in the total cohort (P = 0.1). The severe hypoglycaemia rates were 0.66 ± 1.61 and 0.17 ± 0.42 episodes/patient/year (P < 0.001). In patients with suboptimal metabolic control, HbA1c decreased from 68 ± 12 mmol/mol (8.4 ± 1.1%) to 64 ± 14 mmol/mol (8.0 ± 1.3%) (P = 0.016), with 37.4% of those in this group having an HbA1c ≤ 58 mmol/mol (7.5%) after 5 years. In patients starting CSII due to severe hypoglycaemia the problem was considered resolved in 93%, and in 64% of those starting CSII because of suboptimal glycaemic control, HbA1c improved significantly. CONCLUSIONS: CSII therapy achieves and maintains its efficacy mainly in terms of reducing severe hypoglycaemia. In the whole group of patients, the reduction in HbA1c is transient and disappears after 5 years.

Coherent amplification of attosecond light pulses in the water-window spectral region.

We present a theoretical study on coherent extreme ultraviolet (XUV) attosecond pulse amplification mediated by nonlinear parametric enhanced forward scattering occurring in the interaction of a strong femtosecond infrared (IR) laser pulse combined with a weak attosecond XUV pulse train with an atom. We predict large amplification of XUV radiation when the IR strong pulse and the XUV weak pulse are optimally phased. We study high-order harmonic processes (HHG) in He, He(+) and Ne(++), and show how although the HHG yield is largely affected by the particular atom used as target, nonlinear parametric XUV amplification is only weakly affected. We conclude that XUV nonlinear parametric attosecond pulse amplification can be most efficiently observed by using atoms with a high ionization potential and that the nonlinear amplification is robust at high photon energies where HHG is not efficient, such as in the water-window spectral region.

Genetic dissection of tomato rootstock effects on scion traits under moderate salinity.

KEY MESSAGE: Rootstock HKT1 genotype affected fruit [Na(+)] and non-commercial fruit yield; QTL analysis of rootstock-mediated scion nutrition is a powerful forward genetic approach to identify wild genes for rootstock breeding. The present study approaches the QTL dissection of rootstock effects on a commercial hybrid variety grafted on a population of RILs derived from Solanum pimpinellifolium, genotyped for 4370 segregating SNPs from the SolCAP tomato panel and grown under moderate salinity. Results are compared to those previously obtained under high salinity. The most likely functional candidate genes controlling the scion [Na(+)] were rootstock HKT1;1 and HKT1;2 as it was previously reported for non-grafted genotypes. The higher fruit [Na(+)] found when rootstock genotype was homozygote for SpHKT1 supports the thesis that scion HKT1 is loading Na(+) into the phloem sap in leaves and unloading it in sink organs. A significant increment of small, mostly seedless, fruits was found associated with SlHKT1 homozygous rootstocks. Just grafting increased the incidence of blossom end rot and delayed fruit maturation but there were rootstock RILs that increased commercial fruit yield under moderate salinity. The heritability and number of QTLs involved were lower and different than those found under high salinity. Four large contributing (>17%) rootstock QTLs, controlling the leaf concentrations of B, K, Mg and Mo were detected whose 2 Mbp physical intervals contained B, K, Mg and Mo transporter-coding genes, respectively. Since a minimum of 3 QTLs (two of them coincident with leaf K and Ca QTLs) were also found governing rootstock-mediated soluble-solids content of the fruit under moderate salinity, grafting desirable crop varieties on stress-tolerant rootstocks tenders an opportunity to increase both salt tolerance and quality.

Comparison of two HIV testing strategies in primary care centres: indicator-condition-guided testing vs. testing of those with non-indicator conditions.

OBJECTIVES: The aim of the study was to compare prospectively indicator-condition (IC)-guided testing versus testing of those with non-indicator conditions (NICs) in four primary care centres (PCCs) in Barcelona, Spain. METHODS: From October 2009 to February 2011, patients aged from 18 to 65 years old who attended a PCC for a new herpes zoster infection, seborrhoeic eczema, mononucleosis syndrome or leucopenia/thrombopenia were included in the IC group, and one in every 10 randomly selected patients consulting for other reasons were included in the NIC group. A proportion of patients in each group were offered an HIV test; those who agreed to be tested were given a rapid finger-stick HIV test (€6 per test). Epidemiological and clinical data were collected and analysed. RESULTS: During the study period, 775 patients attended with one of the four selected ICs, while 66,043 patients presented with an NIC. HIV screening was offered to 89 patients with ICs (offer rate 11.5%), of whom 85 agreed to and completed testing (94.4 and 100% acceptance and completion rates, respectively). In the NIC group, an HIV test was offered to 344 persons (offer rate 5.2%), of whom 313 accepted (90.9%) and 304 completed (97.1%) testing. HIV tests were positive in four persons [prevalence 4.7%; 95% confidence interval (CI) 1.3-11.6%] in the IC group and in one person in the NIC group (prevalence 0.3%; 95% CI 0.01-1.82%; P < 0.009). If every eligible person had taken an HIV test, we would have spent €4650 in the IC group and €396,258 in the NIC group, and an estimated 36 (95% CI 25-49) and 198 persons (95% CI 171-227), respectively, would have been diagnosed with HIV infection. The estimated cost per new HIV diagnosis would have been €129 (95% CI €107-153) in the IC group and €2001 (95% CI €1913-2088) in the NIC group. CONCLUSIONS: Although the number of patients included in the study was small and the results should be treated with caution, IC-guided HIV testing, based on four selected ICs, in PCCs seems to be a more feasible and less expensive strategy to improve diagnosis of HIV infection in Spain than a nontargeted HIV testing strategy.

Intermittent hypobaric hypoxia combined with aerobic exercise improves muscle morphofunctional recovery after eccentric exercise to exhaustion in trained rats.

Unaccustomed eccentric exercise leads to muscle morphological and functional alterations, including microvasculature damage, the repair of which is modulated by hypoxia. We present the effects of intermittent hypobaric hypoxia and exercise on recovery from eccentric exercise-induced muscle damage (EEIMD). Soleus muscles from trained rats were excised before (CTRL) and 1, 3, 7, and 14 days after a double session of EEIMD protocol. A recovery treatment consisting of one of the following protocols was applied 1 day after the EEIMD: passive normobaric recovery (PNR), a 4-h daily exposure to passive hypobaric hypoxia at 4,000 m (PHR), or hypobaric hypoxia exposure followed by aerobic exercise (AHR). EEIMD produced an increase in the percentage of abnormal fibers compared with CTRL, and it affected the microvasculature by decreasing capillary density (CD, capillaries per mm2) and the capillary-to-fiber ratio (CF). After 14 days, AHR exhibited CD and CF values similar to those of CTRL animals (789 and 3.30 vs. 746 and 3.06) and significantly higher than PNR (575 and 2.62) and PHR (630 and 2.92). Furthermore, VEGF expression showed a significant 43% increase in AHR when compared with PNR. Moreover, after 14 days, the muscle fibers in AHR had a more oxidative phenotype than the other groups, with significantly smaller cross-sectional areas (AHR, 3,745; PNR, 4,502; and PHR, 4,790 µm2), higher citrate synthase activity (AHR, 14.8; PNR, 13.1; and PHR, 12 µmol·min-1·mg-1) and a significant 27% increment in PGC-1α levels compared with PNR. Our data show that hypoxia combined with exercise attenuates or reverses the morphofunctional alterations induced by EEIMD.NEW & NOTEWORTHY Our study provides new insights into the use of intermittent hypobaric hypoxia combined with exercise as a strategy to recover muscle damage induced by eccentric exercise. We analyzed the effects of hypobaric exposure combined with aerobic exercise on histopathological features of muscle damage, fiber morphofunctionality, capillarization, angiogenesis, and the oxidative capacity of damaged soleus muscle. Most of these parameters were improved after a 2-wk protocol of intermittent hypobaric hypoxia combined with aerobic exercise.

Spatial-temporal variations in rose leaves under water stress conditions studied by chlorophyll fluorescence imaging.

Spatial-temporal changes were examined by imaging chlorophyll (Chl) a fluorescence in four leaf areas, two central and two external of rose plants (Rosa x hybrida) cv. Grand Gala for 9 days, under progressive water stress. New fluorescence parameters based on the lake model have recently been used to determine Q(A) redox state and excitation energy fluxes in order to gain a better understanding of the mechanisms that occur under drought stress. Chlorophyll fluorescence images showed a spatial variation in the leaves. The lower values for F(o), F(M), phi(2), q(P) and q(L) were found in the internal leaf area while higher values of non-photochemical quenching calculated from Stern-Volmer quenching (NPQ) and phi(NPQ). phi(Po) were more homogeneous throughout leaf. Temporal changes were also observed during the experiment, a 10% decrease in relative water content (RWC) (between day 1 and 2), led to a decrease in photochemical quenching and an increase in non-photochemical processes. Chlorophyll fluorescence parameters were more or less constant till day 8. At the end of the experiment (day 9), energy dissipation by downregulation, electron transport and Q(A) redox state, decreased and phi(NO) increased to compensate the change. Chlorophyll fluorescence parameters based on the lake model q(L), phi(NPQ) and phi(NO) have been found more appropriate for estimating the fraction of open centres, the quantum yield of regulated energy dissipation in photosystem II (PSII) and the quantum yield of non-regulated energy dissipation in PSII, respectively. The F(s)/F(o) ratio is strongly correlated with NPQ and phi(NPQ) up to a RWC of 20%. This coincides with a greater decrease in photochemical quenching and non-photochemical quenching and an increase in phi(NO).

Activation of TNF-alpha transcription utilizes distinct MAP kinase pathways in different macrophage populations.

Stimulation of macrophages by lipopolysaccharide (LPS) leads to the rapid activation of MAP kinases (MAPK) and the subsequent induction of cytokine gene expression. We sought to determine whether LPS-inducible cytokine genes were differentially regulated in macrophages derived from different tissues. Our studies revealed that PD98059, an inhibitor of the extracellular-regulated kinase (ERK) pathway, blocked LPS-induced activation of tumor necrosis factor alpha (TNF-alpha) gene expression in a murine cell line derived from alveolar macrophages but not in a nonpulmonary macrophage cell line. These findings were confirmed using primary murine alveolar and peritoneal macrophages. This suggests that the TNF-alpha promoter contains MAPK-dependent and -independent regulatory elements that are used in a cell type-specific manner. We also found that differences in MAPK-regulated signaling were not mediated by NF-KB, LITAF, Egr-1, CREB, or ATF2/ c-Jun. Together, these studies demonstrate that transcriptional activation of the TNF-alpha gene requires the ERK signaling cascade in selected macrophage populations.

A semiquantitative scoring tool to evaluate eccentric exercise-induced muscle damage in trained rats.

Unaccustomed eccentric exercise is a well-documented cause of exercise-induced muscle damage. However, in trained subjects muscle injury involves only light or moderate tissue damage. Since trained rats are widely used as a model for skeletal muscle injury, here we propose a semiquantitative scoring tool to evaluate muscle damage in trained rats. Twenty male Sprague-Dawley rats were trained fortwo weeks following a two-week preconditioning period, and randomly divided into two groups: control rats (CTL; n=5) and rats with eccentric exercise-induced muscle damage (INJ; n=15). Injured rats were sacrificed at three time points: 1, 3 and 7 days post injury (n=5 each). Transverse sections from the right soleus were cut (10 µm) and stained with haematoxylin-eosin. Samples were evaluated by two groups of observers (four researchers experienced in skeletal muscle histopathology and four inexperienced) using the proposed tool, which consisted of six items organised in three domains: abnormal fibre morphology, necrotic/(re)degenerating fibres (muscle fibre domain), endomysial and perimysial infiltration (inflammatory state domain) and endomysium and perimysium distension (interstitial compartment domain). We observed the expected time course in the six evaluated items. Furthermore, agreement among observers was evaluated by measuring the Intraclass Correlation Coefficient (ICC). Within the experienced group, items from the muscle fibre and interstitial compartment domains showed good agreement and the two items from the infiltration compartment domain showed excellent agreement. in conclusion, the proposed tool allowed quick and correct evaluation of light to moderate muscle damage in trained rats with good agreement between observers.

Physical exercise improves brain cortex and cerebellum mitochondrial bioenergetics and alters apoptotic, dynamic and auto(mito)phagy markers.

We here investigate the effects of two exercise modalities (endurance treadmill training-TM and voluntary free-wheel activity-FW) on the brain cortex and cerebellum mitochondrial bioenergetics, permeability transition pore (mPTP), oxidative stress, as well as on proteins involved in mitochondrial biogenesis, apoptosis, and quality control. Eighteen male rats were assigned to sedentary-SED, TM and FW groups. Behavioral alterations and ex vivo brain mitochondrial function endpoints were assessed. Proteins involved in oxidative phosphorylation (OXPHOS, including the adenine nucleotide translocator), oxidative stress markers and regulatory proteins (SIRT3, p66shc, UCP2, carbonyls, MDA, -SH, aconitase, Mn-SOD), as well as proteins involved in mitochondrial biogenesis (PGC1α, TFAM) were evaluated. Apoptotic signaling was measured through quantifying caspase 3, 8 and 9-like activities, Bax, Bcl2, CypD, and cofilin expression. Mitochondrial dynamics (Mfn1/2, OPA1 and DRP1) and auto(mito)phagy (LC3II, Beclin1, Pink1, Parkin, p62)-related proteins were also measured by Western blotting. Only the TM exercise group showed increased spontaneous alternation and exploratory activity. Both exercise regimens improved mitochondrial respiratory activity, increased OXPHOS complexes I, III and V subunits in both brain subareas and decreased oxidative stress markers. Increased resistance to mPTP and decreased apoptotic signaling were observed in the brain cortex from TM and in the cerebellum from TM and FW groups. Also, exercise increased the expression of proteins involved in mitochondrial biogenesis, autophagy and fusion, simultaneous with decreased expression of mitochondrial fission-related protein DRP1. In conclusion, physical exercise improves brain cortex and cerebellum mitochondrial function, decreasing oxidative stress and apoptotic related markers. It is also possible that favorable alterations in mitochondrial biogenesis, dynamics and autophagy signaling induced by exercise contributed to increased mitochondrial plasticity leading to a more robust phenotype.

Exercise modulates liver cellular and mitochondrial proteins related to quality control signaling.

AIMS: The effects of exercise on cardiac and skeletal muscle, including the increase on mitochondrial function, dynamics, biogenesis and autophagy signaling are well described. However, these same effects on liver mitochondria, important in the context of hepatocyte ability to mitigate drug-induced injury and obesity-related disorders, are not fully understood. Therefore, the effects of two distinct chronic exercise models (endurance training--ET and voluntary physical activity--VPA) on liver cellular and mitochondrial quality control were analyzed. MAIN METHODS: Eighteen male-adult Sprague-Dawley rats were divided into sedentary (SED), ET (12-week treadmill) and VPA (12-week voluntary free wheel). Liver mitochondrial alterations were evaluated by semi-quantification of proteins involved in oxidative stress (SIRT3, p66shc, p66(Ser36)), biogenesis (citrate synthase, PGC-1α and mtTFA), dynamics (MFN1, OPA1 and DRP1) and auto(mito)phagy (Beclin-1, Bcl-2, LC3II/LC3I, p62, Parkin and PINK) signaling. Liver ultrastructural alterations were also evaluated. KEY FINDINGS: Both exercise models induced beneficial alterations on liver mitochondrial morphology and increased mitochondrial biogenesis (PGC-1α and mtTFA), autophagy-related proteins (Beclin-1, LC3-II, LC3II/LC3I), and DRP1 and SIRT3 proteins. Increased citrate synthase activity and OPA1, p62 and Parkin content as well as decreased PINK protein levels were only observed after ET. VPA decreased OPA1, Beclin-1/Bcl-2, Parkin and p66(Ser36). Mitochondrial density and circularity increased in both exercised groups. SIGNIFICANCE: Both chronic exercise models increased proteins related with mitochondrial biogenesis and alteration proteins involved in mitochondrial dynamics and autophagy signaling, suggesting that exercise can induce liver mitochondrial adaptive remodeling and hepatocyte renewal.

Molecular and conventional epidemiology of tuberculosis in an inner city district.

SETTING: Molecular epidemiology has underlined the importance of recent tuberculosis (TB) transmission and has uncovered notable discrepancies compared with conventional epidemiology. OBJECTIVES: 1) To determine, by RFLP analysis, the percentage of clustered cases in an inner city district with a high incidence of TB (163/100,000) and the groups at risk of being clustered; and 2) to compare the role of conventional contact tracing (CCT) with that of RFLP. DESIGN: RFLP was carried out using the IS6110 and pTBN12 (PGRS) sequences of 165 cultures positive for Mycobacterium tuberculosis in the Ciutat Vella district of Barcelona during 1997-1998. Contact tracing was carried out in 171 of 251 declared cases (68.1%). Associations were assessed by calculating odds ratios (OR) with 95% confidence intervals (CI). Logistic regression was used for multivariate analysis. RESULTS: Using RFLP, 76 (46.0%) strains were found to be clustered by IS6110 and PGRS. From CCT, 30 new patients were found among 858 contacts (3.5%) and 57 patients were linked. In terms of RFLP and CCT, the main risk factor was intravenous drug use (IVDU). In 44 cases who lived alone and were not involved in CCT, 50% were in RFLP clusters. The concordance rate between RFLP and CCT was 8/13 (61.5%); the disagreement corresponded to a cluster of five recent immigrants from Africa. Subsequent to RFLP, an epidemiological connection was found in 15/55 cases (27.2%). CONCLUSIONS: The percentage of clustered cases is very high. CCT was useful for identifying new cases, but it was insufficient for detecting the pathways of transmission. CCT coverage needs to be improved in marginalized individuals, and the results correlated with those of RFLP.

[Localized hyperthermia of the prostate via the rectum in chronic inoperable urinary retention]. / L'hyperthermie localisée de la prostate par voie rectale dans les rétentions urinaires chroniques inopérables.

Local transrectal hyperthermia of the prostate was used to treat 16 poor surgical risk patients who had an indwelling catheter because of chronic urinary retention caused by benign prostatic hypertrophy. We used the Brucker (Prost-care) model with a rectal applicator, a water cooling system and a microwave generator operating at 915 MHz. Intraprostatic temperatures of 42 degrees to 44 degrees C were measured by a radiometric system. 12 patients received a complete treatment which consists in 6 to 10 sessions of one hour and was controlled at least one month later. 8 of them no longer need a catheter. Uroflowmetry was between 7.5 and 17.6 ml/sec and residual urine appreciate by ultrasounds was less than 60 cm3.

Chronic caffeine or theophylline exposure reduces gamma-aminobutyric acid/benzodiazepine receptor site interactions.

Methylxanthines, such as caffeine and theophylline, are adenosine receptor antagonists that exert dramatic effects upon the behavior of vertebrate animals by increasing attentiveness, anxiety, and convulsive activity. Benzodiazepines, such as flunitrazepam, generally exert behavioral effects that are opposite to those of methylxanthines. We report the finding that chronic exposure of embryonic brain neurons to caffeine or theophylline reduces the ability of gamma-aminobutyric acid (GABA) to potentiate the binding of [3H]flunitrazepam to the GABA/benzodiazepine receptor. This theophylline-induced "uncoupling" of GABA- and benzodiazepine-binding site allosteric interactions is blocked by chloroadenosine, an adenosine receptor agonist, indicating that the chronic effects of theophylline are mediated by a site that resembles an adenosine receptor. We speculate that adverse central nervous system effects of long-term exposure to methylxanthines such as in caffeine-containing beverages or theophylline-containing medications may be exerted by a cell-mediated modification of the GABAA receptor.

[Economic evaluation of a new selective COX-2 NSAID, rofecoxib, in a real practice context]. / Evaluación económica de un nuevo AINE COX-2 selectivo, rofecoxib, en un entorno de práctica real.

OBJECTIVE: To evaluate the economic and health consequences of the use of a new COX-2 anti-inflammatory drug. DESIGN: Cost-effectiveness analysis by modelling three options for prescribing non-steroidal anti-inflammatories (NSAIDs) in patients diagnosed with arthrosis and undergoing long-term NSAID treatment. Option 1: NSAID prescription without gastric protection for low-risk patients, and with gastric protection for patients running a medium or high risk of developing complications (current procedure). Option 2: exclusive prescription of rofecoxib (25 mg/day) for all patients (high and low risk). Option 3: single-therapy rofecoxib (25 mg/day) prescription for patients sensitive to combined treatment (NSAIDs and gastric protection) due to the risk of complications. In each case, resources and expected clinical benefits were accounted for by the calculation of the cost of avoidance of each moderate or high gastro-intestinal side-effect (GISE) (symptomatic ulcer or complication) avoided. The direct economic impact on the centre of passing from option 1 to any of the other two options was calculated. SETTINGS AND PARTICIPANTS: The study, conducted in the Barceloneta Primary Care Centre, included in its modelling the 124 patients diagnosed with arthrosis who received NSAIDs continuously during the year 2000. RESULTS: Option 2 supposed avoiding 1.5 cases of GISE (0.5 symptomatic ulcers and 0.9 serious complications), to an additional cost of 336 566.78 euros. Each case avoided would cost 24,641.50 euros; and each serious case avoided 38,464.79 euros. Option 3 avoided 0.25 cases of serious complication, at an additional cost of 9,015.18 euros. Avoiding one case would cost 37,262.75 euros. CONCLUSIONS: The overall health benefits arising from the universal or partial introduction of a selective COX-2 inhibitor NSAID involve a unit cost of 24,040.48 or 36,060.73 euros, depending on the option. The impact on the Centre s pharmaceutical budget would increase by 1.3% under option 2, and by 0.35% under option 3.

Multiple tumors in the same kidney: incidence and therapeutic implications.

Conservative surgery is proposed by many authors for small kidney tumors. We reviewed retrospectively the pathology reports of 727 operated patients. In 257, tumor diameter was less than or equal to 5 cm, in 158 it was less than or equal to 4 cm, and in 78 it was less than or equal to 3 cm. The number of multiple tumors were 26 (10.1%), 17 (10.75%), and 10 (12.8%) respectively, which means that there is a 10% potential risk of leaving a tumor in situ in case of conservative surgery. There is no security size limit. These parameters have to be considered before proposing conservative surgery for small kidney tumors as a standard treatment.

Chronic agonist exposure induces down-regulation and allosteric uncoupling of the gamma-aminobutyric acid/benzodiazepine receptor complex.

Using [3H]flunitrazepam as a probe for the benzodiazepine-sensitive modulator site located on the gamma-aminobutyric acid (GABA)A receptor complex, we have investigated the cellular regulation of the GABAA receptor in neuronal cultures derived from embryonic chick brain. Treatment of cultures with 1 mM GABA for 48 hr causes a reversible 35% decrease in the number of [3H]flunitrazepam binding sites with no change in affinity. The EC50 for chronic GABA-induced down-regulation is 94 microM and the half-time is 25 hr. The effect of GABA is blocked by SR-95531, a GABAA receptor antagonist, and mimicked by muscimol but not baclofen. Consistent with the decrease in [3H]flunitrazepam binding, chronic GABA exposure causes a 43% decrease in the binding of [35S]t-butylbicyclophosphorothionate, a ligand for the receptor-associated chloride ionophore. In addition to chronic GABA-induced down-regulation, allosteric interactions between GABA and benzodiazepine recognition sites are uncoupled by 34%. The half-time and pharmacology for chronic GABA-induced uncoupling is indistinguishable from that for GABA-induced down-regulation, consistent with the hypothesis that the action of GABA at a common site induces both down-regulation and uncoupling.

gamma-Aminobutyric acid (GABA) induces a receptor-mediated reduction in GABAA receptor alpha subunit messenger RNAs in embryonic chick neurons in culture.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, is known to interact with a subclass of receptors that activate a ligand-gated chloride ion channel. Exposure of cultured embryonic chick neurons to physiological concentrations of GABA results in a time-dependent down-regulation of these GABAA receptors. To delineate the cellular mechanism(s) responsible for agonist-induced down-regulation of GABAA receptors we quantified the levels of GABAA receptor alpha subunit messenger RNAs, which encode the subunit(s) containing agonist recognition site(s), and observed a marked reduction in alpha subunit mRNAs following exposure of embryonic chick neurons to GABA. Both the down-regulation of GABAA receptors and the reduction in alpha subunit mRNAs induced by GABA were completely antagonized by the specific GABAA receptor antagonist SR-95531. These data demonstrate the presence of an agonist-induced receptor-mediated mechanism for regulating the expression of receptor subunit-encoding mRNAs that may be involved in the development of tolerance to the pharmacological actions of drugs known to act via GABAA receptors.

gamma-Aminobutyric acidA receptor regulation in culture: altered allosteric interactions following prolonged exposure to benzodiazepines, barbiturates, and methylxanthines.

In previous reports, we have described the use of primary neuronal cultures derived from chick brain to study the regulation of the gamma-aminobutyric acidA (GABAA) receptor complex. Chronic exposure of cultures to GABA, benzodiazepines, or methylxanthines results in decreased enhancement of [3H]flunitrazepam binding by GABA, consistent with an allosteric uncoupling of GABA and benzodiazepine recognition sites of the GABAA receptor. In the present communication, we extend our studies of the pharmacology of benzodiazepine- and methylxanthine-induced uncoupling of GABA/benzodiazepine recognition site interactions and present evidence to show that certain barbiturates (barbital and pentobarbital) also induce uncoupling. Chronic exposure to flurazepam (a high efficacy benzodiazepine) elicits no change in the number of benzodiazepine binding sites or the affinity of benzodiazepine binding in the absence of GABA. Whereas flurazepam and theophylline decrease coupling, Ro15-1788 (a low efficacy benzodiazepine) inhibits flurazepam-induced but not theophylline-induced uncoupling, suggesting that theophylline and flurazepam act through separate receptors. Flurazepam-induced uncoupling is not prevented by SR-95531 or picrotoxin (specific inhibitors of GABA action) and, therefore, is not an indirect effect mediated by endogenous GABA. The onset of flurazepam-induced uncoupling (EC50 approximately 1 microM) exhibits a t 1/2 of about 18 hr, in general agreement with the half-time for receptor turnover. Uncoupling is reversible following washout and recovery at 37 degrees. These results are discussed in terms of mechanisms of GABAA receptor regulation in response to chronic exposure to functionally homologous or heterologous ligands.